Project Summary Neisseria gonorrhoeae (Gc) and Neisseria meninigitidis (Nm) are pathogenic bacteria causing the sexually transmitted infection gonorrhea and meningococcal meningitis respectively. Gc is a seriously world-wide health concern, especially considering the rise of antibiotic resistance in Gc strains. Nm is a less severe threat, however development of vaccines and antibiotics to prevent and treat Nm infection is still needed. Understanding Neisseria infection and pathogen-host interactions at the basic molecular level becomes crucial for these essential tasks. Opacity-associated (Opa) proteins in Neisseria are known to trigger engulfment of the bacteria into human cells via their interaction with members of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAM or CCM) family. The structure of Opa has been characterized by our lab, but attempts to characterize the Opa-CCM molecular complex have conflicted with the established knowledge of the field. There is evidence that lipooligosaccharide (LOS), a dominant component of the neisserial outer membrane, can bind Opa proteins on adjacent bacteria, and that changes in LOS structure can affect bacterial opacity, Opa expression, Opa-CCM interactions, and Neisseria survival. For these reasons, I hypothesize that lipooligosaccharide is an important factor in Neisseria for maintenance of Opa loop structure, for facilitating Opa-CCM complex formation and for triggering engulfment of Neisseria via Opa-CCM interaction, and may be an essential molecular piece we have been missing in our previous experimental characterization. I propose to build upon the lab?s expertise with Opa and CCM proteins to (i) determine the role of LOS on the Opa protein and the structural assembly of Opa?s characteristic dynamic and hypervariable loops (Aim 1) and (ii) to assess the effect of LOS on the affinity of the Opa-CCM complex formation and its ability to trigger engulfment in human cells in a CCM-dependent manner. I will take a biophysical approach to Aim 1, utilizing spectroscopic techniques to characterize any change in Opa?s conformational heterogeneity or secondary structure in the presence of LOS, which will be a large part of my training. In a world-class environment, I will be trained in electron paramagnetic resonance (EPR) spectroscopic techniques, circular dichroism and nuclear magnetic resonance (NMR) spectroscopy. In Aim 2, I will combine biochemical and cell-based approaches to characterize the effect of LOS on Opa-CCM protein complex formation and cellular uptake. This experimental approach will supplement my biophysical training and help me become a well-rounded scientist who can tackle complex biological questions with an arsenal of techniques. By describing the molecular basis for Opa-CCM interaction, we will provide a more solid foundation for the development of novel vaccines and antibiotics to the broader community studying Neisseria infection.